PROJECT SUMMARY/ABSTRACT The anterior surface of the eye functions as a barrier to the external environment, protects the delicate underlying structures from injury, supports a tear film and maintains transparency. These functions are achieved, in part, through the corneal and limbal epithelia, which must maintain proper proliferation and differentiation. Autophagy, a catabolic process by which cells adapt to intrinsic and extrinsic stress-related situations, has been shown to also regulate proliferation and differentiation. Despite the many advances made in our understanding of corneal and limbal epithelial biology, autophagy remains largely unstudied. We have recently reported that: (i) under resting conditions, autophagy is greater in limbal epithelial basal compared with corneal epithelial basal cells; (ii) autophagy contributes to limbal epithelial proliferative capacity; and (iii) autophagy enables proper activation of transit amplifying (TA) cells following wounding. We also have preliminary evidence that: (i) NUS1 is a novel upstream regulator of autophagy in the limbal epithelium; (ii) blocking autophagy impairs corneal epithelial differentiation and the removal of nuclear material (nucelophagy); and (iii) the receptor tyrosine kinase, EphA2, positively affects corneal epithelial differentiation via regulating end stage autophagy. These observations lead us to hypothesize that autophagy is required to maintain limbal epithelial stem and TA cell homeostasis as well as insure proper corneal epithelial differentiation. We propose to test this hypothesis by focusing on how autophagy: (i) affects limbal epithelial stem cell proliferation; (ii) regulates differentiation in the corneal epithelium; and (iii) is regulated by EphA2 in the corneal epithelium. To accomplish these goals, we will capitalize on our ability to conduct gain- and loss-of-function studies of autophagy-related genes and their key up-stream regulators in complimentary model systems that include cultured human limbal (HLEKs) and corneal (HCEKs) epithelial cells, 3-D raft cultures of limbal and corneal epithelia, and mouse models of impaired autophagy and EphA2 signaling. We will assess the functional consequences of such modulations with a combination of morphogenetic (including Structured Illumination Microscopy and TEM), biochemical, molecular biological, cell biological and physiological approaches. By focusing on autophagy, our proposal represents a new approach to study the regulation of: (i) limbal stem and TA cell proliferation; and (ii) corneal epithelial differentiation and nucelophagy. Such knowledge has clinical applicability as sustaining proliferation is necessary for the proper expansion of cultured HLEKs used in ex vivo transplantation as well as in wound healing. The impact of autophagy and nucleophagy on corneal epithelial differentiation has translational relevance since aberrant differentiation is a feature of diseases of the corneal epithelium (e.g., aniridia, chemical burns) and modulation of autophagy-associated genes are potential treatment modalities, Understanding how autophagy is regulated in corneal epithelium is essential as this tissue is routinely subjected to stress, which requires a proper autophagic response to maintain homeostasis.